Terminalia Chebula-haritaki in Cystic Fibrosis

It’s not what you know; it’s who you know, right? Well…in science, not quite. For seventeen-year-old Madhavi Pulakat Gavini, it was the “what” - her new potential treatment for Pseudomonas infection - that won her 2nd place at the Siemens National Competition and a $50,000 college scholarship to add to her 2006 Intel International Science and Engineering Fair grand prize win. For Madhavi, “who” is an important factor, but in a different way.

Pseudomonas aeruginosa (P. aeruginosa), a bacterium that is common in water, soil and on plants, causes serious or deadly infections in people who are immune compromised. A friend’s aunt, who Madhavi came to know well, had cystic fibrosis (CF). For CF patients, Pseudomonas infection of the lungs is one of their number one killers. “So I looked up everything I could about CF when she told me about it,” says Madhavi. “My project just took off from there.”

Plants vs. BacteriaMadhavi’s grandfather, an Ayurveda traditional medicine practitioner and physician in India, encouraged her to test Ayurveda’s traditional herbal extract treatments against P. aurigenosa. She selected 10 extracts used to treat lung conditions. “I just grew my P. aeruginosa strain on the plates and spotted the extracts onto the plates also to see if any of them could inhibit bacterial growth,” says Madhavi. “A couple had a pretty small zone of inhibition, but one particular extract, Terminalia chebula (Tc), dramatically inhibited the bacteria’s growth.”

“Millions of people suffer from chronic Pseudomonas infections,” says Madhavi. “An antiseptic spray or inhaler could greatly improve their quality of life as well as increase their lifespan.”

Madhavi thought she might have something important, but knew she needed to put aside her extracts for a while. P. aeruginosa, because it can live in and infect such a wide variety of organisms, from plants to humans, has different forms. The “rough” form, named for its appearance on a bacterial plate, is a common strain found in soil, water, and plants. This is the laboratory strain Madhavi used in her initial experiments. But in humans, P. aeruginosa is usually in a “smooth” form, a strain that grows an additional, sticky polysaccharide coat, and often exists in a complex bacterial community called a biofilm. Biofilm-forming strains, with their ability to stick to lung cells and resistance to antibiotics, pose the biggest threat to humans. Madhavi needed to test her extract on a biofilm variant.

Up to this point, Madhavi had been pursuing her project for fun over the summer before her junior year of high school in the biology lab of Dr. Lakshmi Pulakat, her mother and mentor at Mississippi State University. When she started at the Mississippi School for Mathematics and Science (MSMS), she took the project with her. It was Madhavi’s first year at the boarding school, and she was really enjoying the atmosphere. “There are students here who are definitely interested in research, and a lot of them are pretty gifted. So it’s fun,” says Madhavi.

Madhavi enrolled in MSMS’s dual research program that took her to MSU for research during the week, and she worked on weekends and over school breaks too. She had plenty of resources, but she didn’t have one essential piece for her next step – isolating a biofilm-forming Pseudomonas. “I read in a couple of different places that in order to get it to grow biofilm you need what’s called a drip column,” says Madhavi. “It’s rather expensive so I just wanted to see if I could come up with another way of manipulating the bacteria so it will grow the biofilm.” Engineering this new technique became her independent study project in microbiology class.

Fishing for a BiofilmPseudomonas’s biofilm has two main functions: the first is protection, and the second is to allow the bacterium to adhere to smooth surfaces like the epithelial cells in our lungs. The biofilm’s stickiness also makes the cells adhere to plastic. So Madhavi put plastic micropipette tips in cultures of bacteria floating in water and growth medium. The stickier biofilm-forming bacteria grew on the tips, and this allowed Madhavi to “fish” out few biofilm-forming chromoisolates from the “rough” strain.

Now it was time to test Terminalia chebula against biofilm-forming P. aeruginosa. When Madhavi looked at the large zone of inhibition produced by Tc against her new isolate, she knew that she was looking at something big. None of the drugs for Pseudomonas infection currently on the market could do what Madhavi had just accomplished: inhibit both rough and biofilm-forming P. aeruginosa.

Madhavi’s grandfather never got to hear about Tc’s success, he died in the spring of 2005. But she still had another influential “who” to consult in India, her physician and Ayurveda practitioner grandmother. “She would go back and talk to my mother about her project,” says Dr. Pulakat. “She wanted to confirm the fact that Tc was not dangerous.” Madhavi’s grandmother reassured her that the extract has been used as a medicine for thousands of years, and is even safe enough to give to infants. Madhavi, hoping to turn Tc into an effective treatment for P. aeruginosa, set about trying to determine exactly what molecule in the extract was responsible for its action.

Unmasking a MoleculeMadhavi painstakingly determined that Tc retained effectiveness even after 100-fold dilution, after having been sterilized with the extreme heat and pressure of an autoclave, and after passing through a 0.1 micron filter. Then, because all her experiments had tested Tc against new bacterial growth, she pitted it against bacteria that had already been growing for 5 hours. Within 2 hours of Tc treatment, the P. aeruginosa was dead. Tc’s ability to kill bacteria even through a biofilm was confirmed.

Read more about Madhavi in the January/February Medicine and Health Sciences issue of Imagine Magazine, JHU/CTY's magazine for gifted students.

Genetic analysis was next. Madhavi exposed growing cells to Tc, harvested their RNA and compared it to untreated control cell RNA using microarray analysis. When Madhavi finished analyzing the nearly 6,000 microscopic bright gene spots on her chips to determine the levels of RNA for each protein in treated and untreated cells, the reason behind Tc’s effectiveness was clear. Tc-treated bacteria had a reduction in expression of genes necessary for biofilm production, for energy metabolism, and for infecting cells.

Now, Madhavi is working towards developing an inhaler to deliver Tc to the lungs or a spray to deliver Tc to the skin, surfaces, or environment as a way to prevent infection. “I envision Tc extract being used in a multitude of everyday products ranging from household cleaning supplies, to contact lens solutions to toothpaste additive,” said Madhavi in the conclusion to her Siemens competition presentation. She has proved that linking the extract with gold nanoparticles so that they can be tracked in living animals does not compromise its action, and her next step is to test the extract in a mouse cystic fibrosis model. “Millions of people suffer from chronic Pseudomonas infections,” says Madhavi. “Such an antiseptic spray or inhaler could greatly improve their quality of life as well as increase their lifespan.”

If Tc’s promise holds, who knows how many “whos” Madhavi may end up helping.

Madhavi Pulakat Gavini is a senior at the Mississippi School for Mathematics and Science. She enjoys late-night hot chocolate parties in her dorm, has lots of plants, and is a voracious reader. She was a National History Day finalist in 2005 with a paper about Linus Pauling and his stance on nuclear weapons. She intends to pursue biomedical research as a career.Courtesy : Cogito.Org Online community for academically talented youth

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